Mirnas as novel therapeutic adjuvants and biomarkers for the prognosis and treatment of drug resistant breast cancers

ABSTRACT

Disclosed are methods and compositions for using microRNA (miRNA) for treating cancer. The methods and compositions include hsa-miR-204 and homologs of hsa-miR-204.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 61/727,481 filed on Nov. 16, 2012, which ishereby incorporated by reference in its entirety.

DESCRIPTION BACKGROUND OF THE INVENTION

A. Field of the Invention

The invention relates to use of microRNA (miRNA) for treating cancer, aswell as the use of these compositions to treat cancer.

B. Description of Related Art

Approximately 15-20% of all breast cancers are triple negative breastcancers (TNBCs). These cancers typically exhibit distinct, aggressivemetastatic patterns. Prognoses for those receiving a TNBC diagnosis areoften poor, and disproportionate numbers of breast cancer deaths follow.Despite better chemotherapy response rates in early-stages of TNBCdisease, more than sixty percent of TNBC patients developchemoresistance, which leads to early relapse and shortened survival.

miRNAs are small, non-protein encoding RNAs that function widely in generegulation. Messenger RNAs of more than sixty percent of humanprotein-coding genes may be targeted by miRNAs, which are abundant inmany human cell types (Friedman, et al., 2009). The human genome encodesover 1000 miRNAs (more than 1000 Homo sapiens miRNAs are listed inManchester University's miRBase).

SUMMARY OF THE INVENTION

In one instance, there is disclosed a method for reducing growth ofcancer cells, the method comprising administering an effective amount ofa composition comprising hsa-miR-204 or a homolog thereof to a subjecthaving or suspected of having cancer cells reduced or significantlyreduced in expression of hsa-miR-204 or a homolog thereof. In certainaspects, brain-derived neurotrophic factor (BDNF) or ezrin isoverexpressed or significantly overexpressed in the cancer cells of thesubject having or suspected of having cancer cells reduced orsignificantly reduced in expression of hsa-miR-204 or a homolog thereof.

In some embodiments, the subject is a mammal. In certain aspects, thesubject is a human. The subject may have cancer, such as a breastcancer, an ovarian cancer, or a pediatric renal tumor. In certainaspects, the breast cancer cells of the subject are triple negativebreast cancer (TNBC) cells. In certain aspects, cancer cell growth ismetastatic.

In some embodiments, the disclosed method for reducing growth of cancercells further comprises determining the level of hsa-miR-204 or ahomolog thereof in cells of the subject. In related embodiments, thedisclosed method is for identifying a subject likely to benefit fromtherapy for reducing growth of cancer cells, the method comprisingdetermining the level of hsa-miR-204or a homolog thereof in cells of thesubject.

In certain aspects, determining the level of hsa-miR-204 or a homologthereof is by using a quantitative reverse transcription-polymerasechain reaction (qRT-PCR)-based method or an array hybridization-basedmethod. In some aspects, determining the level of hsa-miR-204 or ahomolog thereof is determining the level in a blood sample obtained fromthe subject of hsa-miR-204 or a homolog thereof. In some aspects, thelevel in a blood sample obtained from the subject of hsa-miR-204 or ahomolog thereof is determined to be at a reduced level.

In some embodiments, the composition comprising hsa-miR-204 or a homologthereof is formulated for systemic administration. In certain aspects,this composition is administered by intravenous injection. In certainfurther aspects, the composition comprising hsa-miR-204 or a homologthereof further comprises a lipid-based delivery vehicle or ananoparticle-based delivery vehicle.

In further embodiments, the subject is further administered acomposition comprising a second active agent. In further aspects, thesecond active agent is a cytotoxic chemotherapeutic, a nanobioconjugate,or a miRNA other than hsa-miR-204 or a homolog thereof. In certainfurther aspects, the cytotoxic chemotherapeutic comprises a taxane, andin certain additional further aspects, the taxane is paclitaxel.

In some embodiments that include administration of a compositioncomprising a second active agent, the administration of a compositioncomprising hsa-miR-204 or a homolog thereof is at the same time asadministration of the composition comprising the second active agent. Insome additional embodiments, the administration of a compositioncomprising hsa-miR-204 or a homolog thereof is before or afteradministration of the composition comprising the second active agent. Incertain aspects, the interval of time between administration of thecomposition comprising hsa-miR-204 or a homolog thereof and the secondactive agent may be 1 to 30 days, or it may be 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or morehours or days, or any integer derivable therein.

In certain embodiments that also include administration of a compositioncomprising a second active agent, the administration of compositioncomprising the second active agent is administered before thecomposition comprising hsa-miR-204 or a homolog thereof. In certainfurther aspects that include this administration of a compositioncomprising a second active agent before the composition comprisinghsa-miR-204 or a homolog thereof, cancer cells have become resistant tothe second active agent. In certain additional further aspects, acomposition comprising a second active agent is administered before thecomposition comprising hsa-miR-204 or a homolog thereof, and the secondactive agent is a chemostherapy drug. In certain additional furtheraspects, the second active agent is taxol. In certain additional furtheraspects, the second active agent is paclitaxel.

In some embodiments of the above-noted methods, the compositioncomprising hsa-miR-204 or a homolog thereof is taken from a preparationcomprising a pharmaceutically acceptable carrier. In some aspects, thepreparation further meets pharmacopeial requirements for sterility,pyrogens, and particulate matter or other contaminants, or is formulatedfor systemic administration, or both meets the above-noted pharmacopeialrequirements and is formulated for systemic administration.

Further disclosed is a method for screening for potential sensitizermiRNA that, when inhibited, increases viability of cancer cell linecells cultured in a sub-lethal concentration of therapeutic activeagent, the method comprising: culturing, for a period, cancer cell linecells in a miRNA-inhibitor-containing culture comprising a sub-lethalconcentration of the therapeutic active agent together with an inhibitorof the candidate sensitizer miRNA; culturing, over the same period,cancer cell line cells in a miRNA-inhibitor-lacking culture comprisingthe sub-lethal concentration of the therapeutic active agent but lackingadded inhibitor of the candidate sensitizer miRNA; and determiningviability of the miRNA-inhibitor-containing cell population and themiRNA-inhibitor-lacking cell population after the period, wherein acandidate sensitizer miRNA is identified as a potential sensitizer miRNAif viability of the miRNA-inhibitor-containing cell population (culturedwith therapeutic active agent and candidate sensitizer miRNA inhibitor)is higher or significantly higher than viability of themiRNA-inhibitor-lacking cell population (cultured with therapeuticactive agent but lacking added inhibitor of the candidate sensitizermiRNA).

In certain aspects of this method for screening, the potentialsensitizer miRNA is hsa-miR-204, hsa-miR-185, hsa-miR-211,hsa-miR-367-5p (also called hsa-miR-367*), or hsa-miR-133A, or a homologof any one of these miRNAs. In certain aspects, the sub-lethalconcentration of the therapeutic active agent is a concentration atwhich 80 percent of the cancer cell line cells remain viable afterculture for 72 hours. In certain further aspects, the therapeutic activeagent is taxol and/or other chemotherapy drugs, for example, at asub-lethal concentration of 2 to 8 nM. In certain additional aspects,the cancer cell line cells are TNBC cells.

Further disclosed is a method for screening for potential de-sensitizermiRNA that, when inhibited, decreases viability of cancer cell linecells cultured in a sub-lethal concentration of therapeutic activeagent, the method comprising: culturing, for a period, cancer cell linecells in a miRNA-inhibitor-containing culture comprising a sub-lethalconcentration of the therapeutic active agent together with an inhibitorof the candidate de-sensitizer miRNA; culturing, over the same period,cancer cell line cells in a miRNA-inhibitor-lacking culture comprisingthe sub-lethal concentration of the therapeutic active agent but lackingadded inhibitor of the candidate de-sensitizer miRNA; and determiningviability of the miRNA-inhibitor-containing cell population and themiRNA-inhibitor-lacking cell population after the period, wherein acandidate de-sensitizer miRNA is identified as a potential de-sensitizermiRNA if viability of the miRNA-inhibitor-containing cell population(cultured with therapeutic active agent and candidate de-sensitizermiRNA inhibitor) is significantly lower than viability of themiRNA-inhibitor-lacking cell population (cultured with therapeuticactive agent but lacking added inhibitor of the candidate de-sensitizermiRNA).

In certain aspects of this method for screening, the potentialde-sensitizer miRNA is hsa-miR-129-3p, hsa-miR-296-5p, hsa-miR-216a,hsa-miR-1237, hsa-miR-1915*, hsa-miR-320d, or a homolog of any one ofthese miRNAs. In certain aspects, the sub-lethal concentration of thetherapeutic active agent is a concentration at which 80 percent of thecancer cell line cells remain viable after culture for 72 hours. Incertain further aspects, the therapeutic active agent is taxol and/orother chemotherapy drugs, for example, at a sub-lethal concentration of2 to 8 nM. In certain additional aspects, the cancer cell line cells areTNBC cells.

In some embodiments, a method is disclosed for reducing growth of cancercells comprising administering an effective amount of a compositioncomprising a sensitizer miRNA or a homolog of sensitizer miRNA to asubject having or suspected of having cancer cells reduced orsignificantly reduced in sensitizer miRNA expression. In someembodiments, a method is disclosed for reducing growth of cancer cells,the method comprising administering an effective amount of a compositioncomprising an inhibitor of a de-sensitizer miRNA or an inhibitor of ahomolog of a de-sensitizer miRNA to a subject having or suspected ofhaving cancer cells significantly increased in de-sensitizer miRNAexpression.

In some embodiments, a method is disclosed for identifying a subjectlikely to benefit from therapy for reducing growth of cancer cells, themethod comprising determining the level of a sensitizer miRNA or ahomolog of sensitizer miRNA in cells of the subject. In certain aspects,the sensitizer miRNA of this method is hsa-miR-204, hsa-miR-185,hsa-miR-211, hsa-miR-367-5p (also called hsa-miR-367*), hsa-miR-133A, ora homolog of one of these sensitizer miRNAs.

In some embodiments, a method is disclosed for identifying a subjectlikely to benefit from therapy for reducing growth of cancer cells, themethod comprising determining the level of a de-sensitizer miRNA or ahomolog of de-sensitizer RNA in cells of the subject. In certainaspects, the de-sensitizer miRNA of this method is hsa-miR-129-3p,hsa-miR-296-5p, hsa-miR-216a, hsa-miR-1237, hsa-miR-1915*, hsa-miR-320d,or a homolog of one of these de-sensitizer miRNAs.

Sequences and SEQ ID NOs for stem loop sequences of sensitizer miRNAshsa-miR-204, hsa-miR-185, hsa-miR-211, hsa-miR-367-5p (also calledhsa-miR-367*), and hsa-miR-133A (i.e., for hsa-miR-133A-2), as well asfor de-sensitizer miRNAs hsa-miR-129-3p (i.e., for-3p region ofhsa-miR-129-1), hsa-miR-296-5p, hsa-miR-216a, hsa-miR-1237,hsa-miR-1915*, and hsa-miR-320d are provided in Table 1.

The term “homolog” when used in reference to a miRNA refers to aoligonucleotide that functions like the referenced miRNA, e.g., capableof targeting gene expression like the referenced miRNA. The homologtypically has the level of similarity in properties (e.g., sequence andstructure) with the reference miRNA that would identify it to a personof ordinary skill in the art in view of this disclosure as being ahomolog of the referenced sequence (e.g., using a miRNA homologidentification tool, such as miRNAminer under default/stringentparameters; see, e.g., miRNAminer tool available online atgroups.csail.mit.edu/pag/mirnaminer/; see also Artzi, et al., 2008).

Unless otherwise specified (e.g., in reference to sequence homologies oralignments), percent values expressed herein for compounds are weight byweight and are in relation to the total composition.

The term “significantly” (e.g., when used in reference to, for example:“reduced” or “increased”; “lower” or “higher”; “repressed” or“overexpressed”; “fails to exceed” or “exceeds” or similar-typequalifiers) refers to a level in a referenced or test sample that is atleast of a magnitude greater than the magnitude of the standard error ofthe assay used to assess the characteristic to which the qualifierapplies. In some instances, statistical significance for the referencedor test sample may be associated with a P value of less than 0.25, 0.15,0.10, 0.05, 0.025, 0.01, 0.005, 0.001, 0.0005, or 0.0001.

A pharmaceutically acceptable carrier may include, but is not limitedto: a virus; a liposome; a nanoparticle; or a polymer, and anycombination thereof. Related delivery vehicles may include, but are notlimited to: liposomes, biocompatible polymers, including naturalpolymers and synthetic polymers; lipoproteins; polypeptides;polysaccharides; lipopolysaccharides; artificial viral envelopes;inorganic (including metal) particles; and bacteria, or viruses, such asbaculovirus, adenovirus and retrovirus, bacteriophage, cosmid, orplasmid vectors.

The terms “about” or “approximately” are defined as being close to asunderstood by one of ordinary skill in the art, and in one non-limitingembodiment the terms are defined to be within 10%, preferably within 5%,more preferably within 1%, and most preferably within 0.5%.

The terms “inhibiting,” “reducing,” “treating,” or any variation ofthese terms, includes any measurable decrease or complete inhibition toachieve a desired result. Similarly, the term “effective” means adequateto accomplish a desired, expected, or intended result.

The use of the word “a” or “an” when used in conjunction with the term“comprising” may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.”

The words “comprising” (and any form of comprising, such as “comprise”and “comprises”), “having” (and any form of having, such as “have” and“has”), “including” (and any form of including, such as “includes” and“include”) or “containing” (and any form of containing, such as“contains” and “contain”) are inclusive or open-ended and do not excludeadditional, unrecited elements or method steps. in relation to the totalcomposition.

The compositions and methods for their use can “comprise,” “consistessentially of,” or “consist of” any of the ingredients or stepsdisclosed throughout the specification.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions of the inventioncan be used to achieve methods of the invention.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G. Genomic loss of miR-204 in cancers. FIG. 1A. Highresolution miRNA-CGH (comparative genomic hybridization) on selectedpediatric renal tumors with (left) or without (right) miR-204 deletions.Deletion of genomic loci containing the miRNA is indicated by dottedperpendicular lines. Dots indicate position and value of each probereflecting copy number change, represented in triplicate on the CGHarray. The trend line represents the average value of the triplicateprobe for each tumor. FIG. 1B. Graphs obtained from meta-analysis ofhigh-resolution CGH of ovarian cancers (n=354; obtained from TCGA)representing a subset of cancers with or without deletion. The deletionof genomic loci containing hsa-miR-204 is indicated by dottedperpendicular lines. FIGS. 1C & D. Allelic PCR of hsa-miR-204 genomiclocus in pediatric renal tumors (C) and ovarian cancers (D). The y-axisdisplays log2 transformed relative quantification values. Dotted linesindicate the loss of copy threshold. FIGS. 1E-G. Graphicalrepresentation of qRT-PCR analysis showing levels of miR-204 inpediatric renal tumors (n=38; E), in advanced stage ovarian cancers(n=11; F) and, in breast cancers (n=10; G), when compared to normalmatched control kidney (n=38), normal ovarian tissues (n=5) and normalmatched breast tissues (n=10).

FIGS. 2A-D. Hsa-miR-204 inhibits tumor growth and metastasis. FIG. 2A.Hsa-miR-204 inhibits anchorage-independent growth. Graph depicts thenumber of colonies formed in soft agar wells by HEK-293 cells stablyoverexpressing either scramble or miR-204, further transfected withmiR-204 inhibitor. (*) P<0.05; (**) P<0.01. Results are the average ofthree independent experiments. FIG. 2B. Hsa-miR-204 overexpressioninhibits tumor growth. HEK-293 cells stably overexpressing eitherscramble control or hsa-miR-204. Bar graph representations correspond tomean tumor volume for hsa-miR-204 (n=9) and scramble control (n=9)transfectants. (*) P<0.001. FIG. 2C. Histological analysis of sectionsfrom tumor xenografts overexpressing either scramble (control) orhsa-miR-204 (miR-204). Images in the right panels represent magnifiedviews of boxed regions indicated in the left panels. Tumor invasion incontrol transfectants is reflected by the invasion of tumor into renaltissue. FIG. 2D. Basement membrane matrix invasion assay of MDA-MB-231cells transfected with 75 nM scrambled sequence (control) or hsa-miR-204mimic (miR-204) or hsa-miR-204 mimic transfected cells furthertransfected with hsa-miR-204 inhibitor (miR-204+inhibitor). Bar graphrepresentations correspond to the average number of invaded cellscounted microscopically in five different fields per filter. (***)P<0.001.

FIGS. 3A-3B. Systemic delivery of miR-204 suppresses tumor metastasis.FIG. 3A. Representative lung sections with metastatic foci in neg.control groups. FIG. 3B. No hepatotoxicity in hsa-miR-204 injected mice.Sections of liver from hsa-miR-204 injected mice show no signs ofhepatotoxicity.

FIGS. 4A-H. Hsa-miR-204 regulates expression of BDNF in cancers. FIGS.4A-C. Higher BDNF expression correlates strongly with lower hsa-miR-204expression in multiple cancers. Graphical representation of qRT-PCRanalysis revealing an inverse correlation between hsa-miR-204 and BDNFexpression in pediatric renal tumors (n=38; A), in advanced stageovarian cancers (n=11; B), and in breast cancers (n=10; C), whencompared to normal matched control kidney (n=38), normal ovarian tissues(n=5), and normal matched breast tissues (n=10). FIG. 4D. Schematic ofthe putative miR-204 binding sequence in the BDNF 3′-UTR. FIG. 4E.QRT-PCR analysis of hsa-miR-204 overexpressing cells and cellstransfected with hsa-miR-204 inhibitors using BDNF specific primers.FIG. 4F. Western blot analysis of HEK-293 cells transfected with miR-204mimic using anti-BDNF antibody (1:1000). β-actin was used as a loadingcontrol. Gel photographs are representative of three independentexperiments. FIG. 4G. Graphical representation of band intensitiesquantified using the Total Labs TL100 1D gel analysis software (n=3).BDNF protein level for the control was set to 100.

FIGS. 5A-C. Hsa-miR-204 inhibits tumor cell migration and invasion byaltering AKT/mTOR/Rac1 signaling. FIG. 5A. Has-miR-204 suppressesactivation of AKT and mTOR signaling. HEK-293 cells transfected withhsa-miR-204 were grown in serum-free conditions and subjected to westernblot analysis using antiphospho-Ser⁴⁷³-AKT (1:1000), anti-total-AKT(1:1000), anti-phospho-Ser^(235/236)-S6 (1:1000), antitotal-S6 (1:1000),anti-phospho-Thr^(37/46)-4E-BP1 (1:1000) and anti-total-4E-BP1 (1:1000).β-actin (1:10,000) was used as a loading control. Gel photographs arerepresentative of three independent experiments. FIG. 5B. Hsa-miR-204increases the sensitivity of HEK-293 cells to apoptosis as determined byAnnexin V/PI staining using the FITC-Annexin V Apoptosis Detection Kit.The percentage cell population shown is the mean±SEM of threeindependent experiments. FIG. 5C. Western blot analysis of HEK-293 cellstransfected with hsa-miR-204 using anti-caspase-3 (1:500) antibody andanti-PARP (1:1000) reveals increased cleavage of caspase-3 and PARP.β-actin (1:10,000) was used as a loading control. Gel photograph isrepresentative of three independent experiments.

FIG. 6. Screen in which 974 inhibitors were successfully assayed. Thescreen of MDA-468 cells with paclitaxel (Pac) yielded 47 miRNAssensitizers with a viability ratio (v.r.)<0.85. Of these, 10 miRNAs weresignificant at the p<0.05 level. Applying criteria noted to the screenresulted in the selection of sensitizer and de-sensitizer miRNAs. One ofthese miRNAs, sensitizer hsa-miR-204 (miR-204), when overexpressed,generated significantly reduced viability of MDA-468 cells in thepresence of paclitaxel relative to control.

FIG. 7. Inverse correlation in expression between hsa-miR-204 (miR-2×4)and its target genes BDNF and Ezrin.

FIG. 8. BDNF expression and ezrin expression are bona fide targets ofhsa-miR-204 (miR-2×4).

FIGS. 9A-B. Copy number loss of chromosomal regions containing miRNAs intumors. FIG. 9A. Sample results for various human miRNAs and ovariancancer, breast cancer, and pediatric renal tumors. FIG. 9B. Graphsobtained from meta-analysis of high-resolution CGH of breast cancersrepresenting a subset of cancers with or without deletion. The deletionof genomic loci containing hsa-miR-204 is indicated by dottedperpendicular lines.

FIGS. 10A-B. Hsa-miR-204 targets genes associated with tumorigenesis.FIG. 10A. Log₂ fold change for more than 40 genes including BDNF. FIG.10B. Down-regulated genes by biological functions. Note prevalence ofBDNF among down-regulated genes in various categories of biologicalfunctions.

FIGS. 11A-B. BDNF rescues hsa-miR-204-associated or-induced phenotypes.FIG. 11A. BDNF rescues hsa-miR-204-induced cell [non]migrationphenotype. FIG. 11B. BDNF rescues hsa-miR-204-induced cell [non]invasionphenotype.

FIG. 12. Hsa-miR-204 (miR-204) does not affect TRPM3 levels.

FIG. 13 miR-296-5p inhibition sensitizes paclitaxel response in triplenegative breast cancers.

FIG. 14 miR-296-5p inhibits triple negative breast cell migration andinvasion.

FIG. 15 To further establish the efficacy of these miRNAs in treatingTNBC, we cultured tumor tissues chunks (1 mm3) on the dental sponge. Thetumor implant maintained the architecture of the tumor as shown in toptwo panels.

FIG. 16 Addition of sensitizer miRNAs alone using liposome formulationresulted in killing of tumor cells.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The inventors have demonstrated critical roles for miRNAs in mediatingdrug sensitivity/resistance in triple negative breast cancers (TNBCs)cells. Through high-throughput miRNA/miRNA-inhibitor library screens,the inventors have identified miRNAs that uniquely sensitize drugresistant TNBC cells to paclitaxel, a drug commonly used as a first linetreatment for TNBC conditions. These screens also have revealed thatmiRNAs that sensitize resistant TNBC cells to paclitaxel may be presentat a significantly lower levels in sera of relapsed metastatic TNBCpatients compared to sera from healthy siblings.

In addition, candidate sensitizer miRNA has been shown to havedrastically reduced expression in several cancers and to act as a potenttumor suppressor. Using liposome-based approaches, the inventors haveshown in preclinical mouse tumor models that systemic delivery ofcandidate sensitizer miRNA suppresses breast cancer lung metastasiswithout hepatotoxicity.

The inventors have demonstrated that candidate sensitizer miRNA exertsits function by targeting genes involved in tumorigenesis. These includethe gene encoding brain-derived neurotrophic factor (BDNF), aneurotrophin family member known to promote tumor angiogenesis andinvasiveness. Analysis of primary tumors has revealed that a markedreduction in expression of candidate sensitizer miRNA is accompanied byincreased expression of BDNF or its receptor tropomyosin-related kinaseB (TrkB). These analyses also have revealed that loss of candidatesensitizer miRNA results not only in BDNF overexpression but also inboth subsequent activation of the small GTPase Rac as well as actinreorganization through the AKT/mTOR signaling pathway leading to cancercell migration and invasion.

Among some of the significant specific demonstrations of the inventorsare the following: miRNA-mediated sensitization of drug resistant cancercells by candidate sensitizer miRNA to paclitaxel; suppression of TNBClung metastasis after systemic delivery of candidate sensitizer miRNA;and lower levels (potentially significant for diagnostic purposes) ofcandidate sensitizer miRNA in TNBC patient sera. Identification ofmiRNAs that confer drug sensitivity may help provide a new generation oftherapeutics, as well as provide new prognostic tools to monitor TNBCtreatment responses to specific drugs through analysis of miRNAexpression profiles of TNBC cells. In addition, because paclitaxel isused in treating many types of cancer, the invention has beneficialimplications not simply for breast cancer treatment but also fortreatment of many other types of cancer.

The inventors have demonstrated that genomic loss of tumor suppressorhsa-miRNA-204 promotes cancer cell migration and invasion by activatingAKT/mTOR/Rac1 signaling and actin reorganization.

Chromosomal regions containing microRNAs may be functionally importantin cancers. The inventors have demonstrated that genomic loci encodinghsa-miR-204 are frequently lost in multiple cancers, including ovariancancers, pediatric renal tumors, and breast cancers. In particular,hsa-miR-204 has been shown to have drastically reduced expression inseveral cancers and acts as a potent tumor suppressor, inhibiting tumormetastasis in vivo when systemically delivered.

The inventors have demonstrated that hsa-miR-204 exerts its function bytargeting genes involved in tumorigenesis, including brain-derivedneurotrophic factor (BDNF)-a neurotrophin family member which is knownto promote tumor angiogenesis and invasiveness. Analysis of primarytumors further has revealed that increased expression of BDNF or itsreceptor tropomyosin-related kinase B (TrkB) parallel a markedly reducedexpression of hsa-miR-204. Loss of hsa-miR-204 results in BDNFoverexpression and subsequent activation of the small GTPase Rac as wellas actin reorganization through the AKT/mTOR signaling pathway leadingto cancer cell migration and invasion.

The microdeletion of genomic loci containing hsa-miR-204 is directlylinked with the deregulation of key oncogenic pathways that providecrucial stimulus for tumor growth and metastasis. Accordingly, theinventors therapeutically manipulated hsa-miR-204 levels in order tosuppress tumor metastasis.

Sequences for hsa-miR-204 and other miRNAs identified herein areprovided in Table 1.

TABLE 1 miRBase Accession Nos., SEQ ID NOS., and Sequences of SelectmiRNAs: second- & fourth-row subsequences for each miRNA are-5p & -3p subsequences, respectively, where applicable. miRBaseMI000---- SEQ miRNA Acc. No. ID NOS. SEQUENCE hsa-miR- 0284 1GGCUACAGUCUUUCUUCAUGUGACUCGUGGAC 204 2 UUCCCUUUGUCAUCCUAUGCCU 3GAGAAUAUAUGAAGGAG 4 GCUGGGAAGGCAAAGGGACGU 5 UCAAUUGUCAUCACUGGC hsa-miR-0482 6 AGGGGGCGAGGGAU 185 7 UGGAGAGAAAGGCAGUUCCUGA 8 UGGUCCCCUCCCC 9AGGGGCUGGCUUUCCUCUGGUC 10 CUUCCCUCCCA hsa-miR- 0287 11UCACCUGGCCAUGUGACUUGUGGGC 211 12 UUCCCUUUGUCAUCCUUCGCCU 13AGGGCUCUGAGCAGG 14 GCAGGGACAGCAAAGGGGUGC 15 UCAGUUGUCACUUCCCACAGCACGGAGhsa-miR- 0775 16 CCAUU 367 17 ACUGUUGCUAAUAUGCAACUCU 18 GUUGAAUAUAAAUUGG19 AAUUGCACUUUAGCAAUGGUGA 20 UGG hsa-miR- 0451 21 GGGAGCCAAAUGCUUUGCUAG133A-2 22 AGCUGGUAAAAUGGAACCAAAU 23 CGACUGUCCAAUGGA 24UUUGGUCCCCUUCAACCAGCUG 25 UAGCUGUGCAUUGAUGGCGCCG hsa-miR- 0252 26 GGAU129-1 27 CUUUUUGCGGUCUGGGCUUGC 28 UGUUCCUCUCAACAGUAGUCAGG 29AAGCCCUUACCCCAAAAAGUAU 30 CU hsa-miR- 0747 31 AGGACCCUUCCAG 296 32AGGGCCCCCCCUCAAUCCUGU 33 UGUGCCUAAUUCA 34 GAGGGUUGGGUGGAGGCUCUCC 35UGAAGGGCUCU hsa-miR- 0292 36 GAUGGCUGUGAGUUGGCU 216a 37UAAUCUCAGCUGGCAACUGUGA 38 GAUGUUC 39 UCACAGUGGUCUCUGGGAUUAU -- --hsa-miR- 6327 40 GUGGGAGGGCCCAGGCGCGGGCAGGGGUGGG 1237 GGUGGCAGAGCGCUGUCC41 CGGGGGCGGGGCCGAAGCGCG 42 GCGACCGUAAC 43 UCCUUCUGCUCCGUCCCCCAG -- --hsa-miR- 8336 44 UGAGAGGCCGC 1915* 45 ACCUUGCCUUGCUGCCCGGGCC 46GUGCACCCGUGGG 47 CCCCAGGGCGACGCGGCGGG 48 GGCGGCCCUAGCGA hsa-miR- 8190 49UUCUCGUCCCAGUUCUUCCCAAAGUUGAG 320d-1 50 AAAAGCUGGGUUGAGAGGA -- -- -- ---- --

A. MicroRNAs and Cancer.

Identification of chromosomal regions harboring oncogenes and tumorsuppressor genes has led to better understanding of the tumorpathogenesis and better treatment outcomes (Bayani, et al., 2007).Although several cancer-related genes have been identified in regionswith chromosomal abnormalities (Albertson, et al., 2003), additionalregions with random or recurrent chromosomal abnormalities harboringfactors important in cancer growth and progression remain to beidentified. Recent studies have indicated that microRNAs (miRNAs) areone such group of factors (Varambally, et al., 2008). MiRNAs are small,endogenous, non-coding RNAs that regulate post-transcriptional geneexpression by binding to 3′ untranslated (UTR) regions of target mRNAs.MiRNAs are known to have important functions in multiple biologicalprocesses including development and differentiation (Bartel, et al.,2004). Deregulated expression of miRNAs has been implicated in severalhuman diseases including cancer (Bartels, et al., 2009). Evidencesuggests that miRNAs can act as oncogenes or tumor suppressor genes(Esquela-Kerscher, et al., 2006).

Hsa-miR-204 has been identified as often being lost in multiple cancers.Results of studies by the inventors have demonstrated that hsa-miR-204acts as a potent tumor suppressor by suppressing the function of genesassociated with tumorigenesis, including brain-derived neurotrophicfactor (BDNF). BDNF is a physiologically important nerve growth factorthat plays a critical role in the development of nervous system bybinding and subsequently activating the tyrosine kinase receptor,tropomyosin-related kinase B (TrkB) (Lewin, 1996; Segal, et al., 1996).In addition, BDNF/TrkB pathway is reported to have a critical role intumorigenesis as it promotes proliferation, differentiation,angiogenesis and tumor invasiveness (Au, et al., 2009). Overexpressionof BDNF/TrkB has also been implicated in poor prognosis of several solidtumors including neuroblastoma, ovarian, breast, prostate and lungcancers (Brodeur, 2003; Edsjo, et al., 2003; Nakagawara, et al., 1994).Loss of hsa-miR-204 results in BDNF/TrkB overexpression and concomitantactivation of AKT/mTOR/Rac1 signaling pathway leading to actinreorganization during cancer cell migration and invasion. These resultsunderline that chromosomal regions containing specific miRNAs may haveparticular functional importance in tumorigenesis.

B. Somatic Loss of Hsa-miR-204 in Cancers.

To identify miRNAs that are associated with aberrant chromosomal regionsin human cancers, high-resolution custom miRNA comparative genomichybridization (CGH) was used together with high density CGH publicdomain datasets for ovarian cancers, breast cancers and pediatric renaltumors. This analysis revealed several miRNAs in the minimal chromosomaldeletion and amplification regions of these tumors. To begin tounderstand the functional importance of chromosomal loci associated withmiRNAs in tumorigenesis, miRNAs exhibiting genomic loss were examined(FIG. 1 and, for example, FIG. 9A). The 9q21.12 chromosomal regioncontaining hsa-miR-204 was frequently lost in 44.63% (158/354) ofovarian cancers, 28% (10/35) of breast cancers, and 40% (15/38) ofpediatric renal tumors (concerning pediatric renal tumors and ovariancancers, see also respectively FIGS. 1A and 1B; for breast cancers, seeFIG. 9B), which is further verified by quantitative genomic real-timePCR analysis (FIGS. 1C and 1D). Furthermore, reduction in the levels ofmature miR-204 also strongly correlated with its genomic DNA content inall three tumor types (FIGS. 1E, 1F and 1G).

C. Hsa-miR-204 Acts as a Potent Tumor Growth and Metastasis Suppressor.

The drastically reduced expression of hsa-miR-204 in multiple cancertissues prompted addressing the role of hsa-miR-204 in tumorigenesis.The effect of hsa-miR-204 on anchorage independent growth was firstaddressed. HEK-293 cells passaged over 52 times are reported to behighly tumorigenic (Shen, et al., 2008), and embryonal kidney HEK-293cells overexpressing hsa-miR-204 exhibited reduced colony-formingcapacity. But colony-forming capacity was rescued with theoverexpression of an inhibitor of hsa-miR-204 (FIG. 2A). Similar resultswere also obtained with hsa-miR-204 overexpression in breast cancerMDA-MB-231 cells and ovarian cancer SKOV3 cells (data not shown).Further to confirm the potential tumor suppressor-like activity ofhsa-miR-204, high-passage HEK-293 cells stably overexpressing eitherhsa-miR-204 or a scrambled sequence were injected into the kidneycapsules of nude mice and tumor growth and metastasis were evaluated 24days after injection. In sharp contrast to control tumors, hsa-miR-204overexpressing tumors were dramatically reduced in size. Whetherhsa-miR-204 also inhibits tumor cell invasion was next examined.Histological analysis of xenograft tumor sections indicated drasticallydecreased or no invasiveness of tumors into renal tissues thatoverexpressed miR-204 compared to control (FIG. 2C). Consistent withthis, hsa-miR-204 overexpression drastically reduced the migratory andinvasive capabilities of breast cancer (MDA-MB-231) and ovarian cancer(SKOV3) cells in vitro (FIG. 2D; and data not shown).

D. Therapeutic Targeting of Hsa-miR-204.

To further substantiate the metastasis suppressor activity ofhsa-miR-204 , therapeutic experiments in a breast cancer-lung metastasismodel were performed. Lung metastases were first established by tailvein injection of MDA-MB-231 breast cancer cells expressingluciferase-GFP. Subsequently, hsa-miR-204 or hsa-miR-204 mutant oligo(negative control) were injected into the tail vein of these nude miceevery five days for 30 days using a LANCErII lipid-based in vivodelivery vehicle. Interestingly, systemic delivery of hsa-miR-204resulted in significant reduction or elimination of lung metastases,while, in contrast, hsa-miR-204 mutant oligo injected mice hadmetastasis including severe lung metastasis (FIG. 3A). In particular,mice injected with hsa-miR-204 displayed no obvious side effects atleast as revealed by the absence of hepatotoxicity or a change in thebody weight (FIG. 3B; and data not shown). These results indicate thathsa-miR204 and its homologs can form the basis for safe and viabletherapeutic regimens to treat tumor growth and metastasis.

In similar experiments, mice were transplanted with triple negativebreast cancer cells and two groups of mice were injected via tail veineither with miR-204 oligos or oligos with seed sequence mutated. Tailvein injection were given every 6 days for 30-days. Interestingly,systemic delivery of miR-2×4 resulted in significant reduction orelimination of lung metastases, while miR-2×4 mutant oligo injected micehad severe lung metastasis. These results indicate that miR-204 can betherapeutically targeted to treat tumor growth and metastasis

E. Hsa-miR-204 Targets Genes Associated with Tumorigenesis.

To understand the mechanism undergirding the role of hsa-miR-204 mayplay in tumorigenesis, genes regulated by miR-204 were identified.Because most miRNAs act to decrease target mRNA levels (Guo, et al.,2010), gene expression analyses on cells overexpressing hsa-miR-204 wereperformed and potential targets of hsa-miR-204 were determined. Of thegenes altered in hsa-miR-204 overexpressing cells, downregulated genesare likely to be directly targeted by hsa-miR-204. Interestingly,several of the downregulated genes are associated with cancer-relatedprocesses and physically or functionally interact with each other, asrevealed by pathway-based analyses (FIGS. 10A and 10B). Of these genes,detailed analyses were performed on one such gene: BDNF, which showedhigher levels of alteration in microarray analysis and was featured inall predicted biological pathways with highest functional enrichmentsignificance. BDNF, with its receptor TrkB, is known play a criticalrole in tumor angiogenesis and metastasis (Edsjo, et al., 2003;Nakagawara, et al., 1994). Moreover, multiple target predictionalgorithms, including Sv/MicrO (Liu, et al., 2010), Bayesian decisionfusion approach (Yue, et al., 2010) and miRmate (Du, et al., 2009) thatthe inventors generated, as well as TargetScan (Lewis, et al., 2005) andPictar (Krek, et al., 2005), also predicted BDNF to be targeted byhsa-miR-204. Furthermore, the hsa-miR-204 binding site was found to beevolutionarily conserved throughout vertebrates, suggesting that it hasan important regulatory function across a variety of species.Importantly, hsa-miR-204 and BDNF/TrkB expression showed a stronginverse correlation in several tumors (FIGS. 4A, 4B, and 4C).

To validate microarray and target prediction results, whetherhsa-miR-204 targets BDNF by binding to the predicted site in its 3′ UTRwas first investigated (FIG. 4 D). Indeed, luciferase activity of apMIR-reporter construct containing the BDNF 3′-UTR was significantlyrepressed, which was further reduced in cells overexpressing hsa-miR-204when compared to the construct without the 3′-UTR. In contrast, mutationof the seed sequence in the BDNF 3′-UTR-containing construct not onlyrestored luciferase activity to near that of the wildtype construct butalso rendered transcripts from the mutant construct insensitive tohsa-miR-204 overexpression, confirming a specific interaction betweenhsa-miR-204 and the predicted binding site in the BDNF 3′-UTR.

To further substantiate these results, the levels of BDNF in cellsoverexpressing hsa-miR-204 were determined. MiR-204 overexpressionresulted in significant reduction of BDNF both at the RNA and proteinlevels (FIGS. 4E, 4F, and 4G). Next, whether or not BDNF is afunctionally important target of miR-204 was examined. To address this,rescue experiments were performed. Reintroduction of BDNF rescuedhsa-miR-204 induced phenotypes including anchorage-independent growth,cell migration and invasion (FIGS. 11A & 11B; and data not shown). Theseresults suggest that hsa-miR-204-meditated regulation of BDNF is animportant event in cancer cell growth, migration, and invasion.

F. Loss of Hsa-miR-204 Activates AKT/mTOR Signaling and Rac1Translocation in Cancer Cells.

To determine the mechanism by which hsa-miR-204 may exhibit its tumorgrowth and metastasis suppressor activity, the effect of hsa-miR-204 onAKT/mTOR signaling was examined in light of BDNF having been shownpreviously to activate AKT pathway (Troca-Martin, et al., 2010); inaddition, selective activation of AKT by mTOR has been shown to regulatecancer cell migration and invasion. Interestingly, hsa-miR-204overexpression resulted in reduced activity of AKT and mTOR downstreamtargets 4E-BP1 and S6 (FIG. 5A). 4E-BP1 is a translation inhibitor thatdissociates from eIF4E upon phosphorylation to allow proteintranslation, and S6 is a ribosomal protein whose phosphorylationfacilitates assembly of the ribosome and consequent translation of mRNA.AKT controls cell invasiveness by regulating multiple processes that areinvolved in actin organization, cell-to-cell adhesion, and cell motility(Kim, et al., 2011; Grille, et al., 2003; Enomoto, et al., 2005).

To examine whether miR-204 may play a direct role in this process, theeffect of hsa-miR-204 overexpression on the activation of the small Gprotein Rac1 was assessed. The small G protein Rac1 functionallyinteracts with AKT/mTOR and is reported to play an important role incell migration and actin reorganization upon induction with BDNF(zadran, et al., 2010) or epidermal growth factor (EGF) (Kim, et al.,2011). Stimulation of MDA-MB-231 cells (and SKOV3 or HEK-293 cells) withBDNF or EGF induced membrane ruffling, and this stimulation caused Rac1to be translocated to the ruffling region (data not shown). In contrast,membrane ruffling and Rac1 translocation were not observed in cellsoverexpressing hsa-miR-204 when induced with BDNF or EGF (data notshown). Evasion of apoptosis, which is critical for tumor growth andprogression (Hanahan, et al., 2000) could be another mechanism centralto oncogenesis in cancers exhibiting loss of hsa-miR-204. Moreover,increased AKT/mTOR activity has also been associated with decreasedapoptosis in cancers (Krishnan, et al., 2006). Indeed, hsa-miR-204overexpression caused a significant increase in the levels of bothactivated caspase-3 and polyADP ribose polymerase (PARP), indicators ofirreversible damage to the integrity of the cell and genome, with aresultant increase in apoptotic activity (FIGS. 5B & 5C). Takentogether, these findings suggest that loss of hsa-miR-204 promotes tumorcell growth, migration and invasion by activating its target genes thatare known regulators of oncogenic signaling cascade.

G. High-throughput Functional Screening.

Hsa-miR-204 is seen to be not only a tumor suppressor but also a miRNAthat sensitizes drug resistant breast cancers to the chemotherapy drugpaclitaxel.

To identify miRNAs that may sensitize paclitaxel resistant TNBC cells topaclitaxel, an unbiased and comprehensive approach was taken: a libraryof 974 chemically synthesized miRNA inhibitors was used in ahigh-throughput screening platform both to identify miRNAs that reduceTNBC cell viability in the presence of a sub-lethal dose of paclitaxel(2 to 8 nM), a dose at which 80% of TNBC cells were still viable, and tovalidate selected candidates. TNBC cell line MDA-468 was used.

The inhibitors, each of which targets one of 974 human microRNAs, werearrayed in a one-inhibitor-one-well format on 96-well micro-titerplates. Transfections of paclitaxel-resistant MDA-468 cells wereperformed in sextuplicate for triplicate analysis in the presence(sub-lethal concentration) and the absence of drug. Cells were subjectedto a 72-hour exposure to drug at or below the IC₂₀, as derived from5-day MTS assays. Cell viability was measured using CellTiter-GloViability Assay (Promega).

Raw values were normalized to internal reference control samples(positive and negative controls) on each plate to permit plate-to-platecomparisons. Each miRNA inhibitor is assigned a viabilityratio-calculated as mean viability in paclitaxel divided by meanviability in the absence of drug (meanpac/meancarrier). In ahigh-throughput screen, the number of statistical comparisonssignificantly exceeds that of the biological replicates. For each miRNAinhibitor, a two sample t-test (with pooled variance) was performed todetermine whether there is a significant difference between the meanvalues under the two experimental conditions (presence and absence ofdrug). Resulting raw p values were inflated based on their rank in thedistribution of all p-values-based on the Benjamini-Hochberg method ofcontrolling the false discovery rate (FDR). This protocol has beenapplied successfully in a genome-wide RNAi-based synthetic lethalscreening study, where it identified a highly reproducible list of hits18 and in the current screens (FIG. 6).

This screen resulted in identification of different categories ofmiRNAs: (1) De-sensitizers: miRNAs that, when inhibited, decreased cellviability (indicating that, when overexpressed, these miRNAs willsupport cell viability) in the presence of paclitaxel when compared tocarrier; (2) Sensitizers: miRNAs that, when inhibited, increased cellviability (indicating that, when overexpressed, these miRNAs willdecrease cell viability) in the presence of paclitaxel when compared tocarrier; and (3) Drug-neutral: miRNAs that, when inhibited, had nosignificant effect on cell viability when compared between paclitaxeland carrier.

One sensitizer, hsa-miR-204, when inhibited, showed the highestmagnitude of an increase in cell viability in paclitaxel-treated TNBCcells when compared to untreated carrier-maintained cells. As shown inFIG. 6, overexpression of sensitizer hsa-miR-204 resulted in asignificant (more than approximately four-fold; P=0.006) decrease inTNBC cell viability in the presence of sub-lethal dose of paclitaxelwhen compared with untreated (carrier) control.

H. Ezrin

The inventors have also identified hsa-miR-204 binding to ezrin RNA, andspecifically binding of the sequence 3′-UUUCCCUU-5′ in the seed area ofhsa-miR-204 to the corresponding 5′-AAAGGGAA-3′ sequence in the ezrin3′-UTR region. Ezrin is a member of the ezrin/radixin/ moesin (ERM)family, and it promotes cytoskeletal reorganization by couplingfunctions of the plasma membrane and actin cytoskeleton of the cell.Ezrin has been implicated in tumor growth and metastasis of severaladult and pediatric tumors, and strong multifocal expression of ezrinhas been associated with poor prognoses for several tumors.P-glycoprotein binds to ezrin at amino acid residues 149 through 242 inthe ferm domain and plays a key role in the multi-drug resistance ofhuman osteosarcoma.

As is the case for the inverse correlation in expression of hsa-miR-204and BDNF loci (FIGS. 4A, 4B, 4C, 4E, 4F, and 4G), there is an inversecorrelation between expression of hsa-miR-204 and ezrin loci (FIG. 7).As is the case for BDNF expression, ezrin expression is a bona fidetarget of hsa-miR-204 (FIG. 8).

I. Additional Considerations

Many miRNAs associated with cancers are known to be localized to genomicfragile sites (Calin, et al., 2004). However, surprisingly little isknown about the functional importance of regions with chromosomalaberrations containing miRNAs. It is likely that initial genetic screensthat aimed at discovering chromosomal abnormalities (with typicallylower comparative genomic hybridization resolution) overlooked changesin genomic regions containing miRNAs. Because miRNAs influence severalgenes in one or more pathways that regulate cell growth and apoptosisand contribute to tumor formation when deregulated, a closer scrutiny ofsmaller genomic regions encoding miRNAs may provide important insightsinto the mechanism of tumorigenesis. Most miRNAs are proposed to bedownregulated in cancers, and microdeletion of genetic regionscontaining specific miRNAs may be an occurrence that plays an importantrole in the development and progression of human cancers.

Evidence is provided herein that hsa-miR-204, which acts as a potenttumor growth and metastasis suppressor, is somatically lost in somehuman cancers. That hsa-miR-204 regulates the expression and function ofthe pro-angiogenic protein BDNF and its receptor TrkB in tumors isdemonstrated, as well as that loss of hsa-miR-204 promotes BDNF (orEGF)-induced cancer cell migration and invasion by activating AKT/mTORpathway leading to Rac1 translocation and actin reorganization. BecauseAKT and Rac1 require each other for their activation (Kim, et al., 2011;Higuchi, et al., 2001), these findings suggest that loss of hsa-miR-204expression in human tumors may induce the positive feedback loop betweenBDNF/AKT1 and Rac1 during growth factor-induced cancer cell migrationand invasion. In support of this, EGF has been shown to activate AKT andfacilitate Rac1 translocation to the cell membrane, and crosstalkbetween EGFR and TrkB has been shown to induce cancer cell migration(Qiu, et al., 2006). Moreover, response to EGF is a key step duringcancer cell invasion and is directly linked with metastasis (Wyckoff, etal., 2004). In addition to cancer cell migration and invasion,activation of BDNF/TrkB signaling may also contribute to evasion ofapoptosis in hsa-miR-204 depleted cells as BDNF/TrkB overexpression hasbeen linked with stabilization and activation of AKT resulting indecreased apoptosis (Siu, et al., 2009). Consistent with this, theseresults demonstrate that hsa-miR-204 overexpression results in reducedphosphorylation and activation of mTOR downstream targets 4E-BP1 and S6kinase.

Because hsa-miR-204 resides within the transient receptor potentialcation channel member 3 (TRPM3) gene, which is a member of transientreceptor potential channels family of proteins, and these proteins areknown to be important for cellular calcium signaling and homeostasis,the speculation is tempting that the phenotypes associated with thegenomic loss of chromosomal loci containing hsa-miR-204 may also becontributed by the host gene TRPM3. But results herein showingsuppression of tumor growth and metastasis following introduction ofhsa-miR-204 alone, with no change in the levels of TRPM3 gene inhsa-miR-204 mimic or inhibitor overexpressing cancer cells (FIG. 12).This clearly suggests that the hsa-miR-204-associated tumor suppressorphenotype is not mediated through TRPM3. However, the possibility cannotbe excluded that hsa-miR-204 and its host gene TRPM3 act synergisticallyto regulate suppression of tumor growth as well as of tumor cellmigration and invasion. Because a definite role for TRPM3 intumorigenesis has not been reported, a detailed study examining TRPM3′spotential tumor suppressor role and its synergistic effects withhsa-miR-204 (if any) may be subjects of future investigations.

Taken together, the findings herein suggest that genetic loci containingspecific miRNA may play a causal role in cancer growth and metastasis byregulating key oncogenic pathways. The ability of hsa-miR-204 to targetBDNF/TrkB, which have significant roles in normal cellular processes andare overexpressed in aggressive cancers, indicates that hsa-miR-204 maycontrol key regulatory mechanisms, and that the dysregulation of suchcontrol can predispose normal developmental/differentiation events toundergo transformation. Therefore, strategies aimed at using hsa-miR-204in therapeutic regimens will have the advantage of reversinginappropriately activated steps in cancer cells to a more normal state.The identification herein of hsa-miR-204 as a potent tumorsuppressor-along with demonstration herein of its therapeutic potentialand negligible hepatotoxicite-establishes a strong rationale fordeveloping hsa-miR-204 as a key component of a viable therapeuticregimen to treat cancers.

EXAMPLES

The following examples are included to demonstrate certain non-limitingaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Many of the following examples provide additional detail on exemplarymaterials and methods.

EXAMPLE 1 Normal and Tumor Tissue Samples and Cell Lines

Cell lines representing pediatric renal tumors (HEK-293; embryonalkidney cell line), ovarian cancer (SKOV3) and breast cancer (MDA-MB-231)used for experiments were cultured according to ATCC protocols.Seventy-six pediatric renal tumors and normal matched kidney wereacquired from the Children's Oncology Group (Arcadia, Calif.). Elevenadvanced stage ovarian tumors and five normal ovarian tissues wereobtained from UT MD Anderson Cancer Center (Houston, Tex.). Fifteenbreast cancer tissues and normal matched tissues were obtained from UTHealth Science Center (San Antonio, Tex.).

EXAMPLE 2 RNA and Protein Analysis

Total RNA was extracted from tumors and normal tissues, as well as allcell lines, and was subjected to qRT-PCR analysis, as describedpreviously (Imam, et al., 2010). Western blot analysis was performed asdescribed previously (Imam, et al., 2010). Antibodies to β-actin(AC-74-A5316) and α-tubulin (T6199) were purchased from Sigma Aldrich.Antibody to BDNF was purchased from Santa Cruz, other antibodiesincluding antibodies specific for caspase-8 (IC12-9746), caspase-3(8G10-9665), caspase-9 (9502), PARP (9542S), AKT (9272), phospho-AKT(9271), S6 ribosomal protein (2217), and phospho-S6 ribosomal protein(5G10-2211S) were purchased from Cell Signaling.

EXAMPLE 3 Genomic PCR Assay

Genomic DNA from pediatric renal tumors (n=38), and advanced stage (IIIand IV) ovarian cancers and matched normal renal (n=38) and ovarian(n=4) tissues were isolated with the DNeasy Genomic DNA Extraction Kit(Qiagen). For genomic analysis of hsa-miR-204, the 2 ^(ΔΔct) method wasadapted using SYBR Green-based quantitative PCR (qRT-PCR) as described(Varambally, et al., 2008). Briefly, 25 ng of genomic DNA was used astemplate to amplify the hsa-miR-204 locus. A representative controltissue sample was used in every assay as a calibrator to which everysample was compared, to obtain a relative quantitation (RQ) value.Genomic DNA from a normal male sample (1x) and a normal female sample(2x) (Promega) was used to compare the levels of phosphoglycerate kinase1 (PGK1) and five X-chromosome specific miRNAs-hsa-miR-424, hsa-miR-503,hsa-miR-766, hsa-miR-222 and hsa-miR-221-to calibrate the extent of lossin the various miRNA loci. RQ values for these regions in male genomicDNA were assessed using the non-X-chromosome TATA binding protein (TBP)gene as the reference. Based on its ability to separate two distinctdata populations, an RQ value of 0.5 and below was considered as loss ofat least one copy of a genomic locus.

Example 4 High-Resolution miRNA Comparative Genomic Hybridization (CGH)Assay

MiRNA CGH analysis was performed on the genomic DNA isolated frompediatric renal tumor samples using an Agilent custom-designedmicroarray as per the manufacturer's protocol. Custom arrays weredesigned based on the Agilent 2×105K Human Whole Genome GenomicMicroarray using Agilent's eArray program (available online atearray.chem.agilent.com/earray), with additional probes that cover allmiRNA regions (200 by before, within and after of each miRNA frommiRBase v13, with triplicate probes to enhance reliability). Array CGHdata analysis was performed with Nexus Copy Number (BioDiscovery) forDNA alteration quantification. Upon determination of samples with orwithout copy number alteration at specific miRNA sites, all array CGHdata was loaded into MATLAB for a composite graphical display. Array CGHshowing position of the probes and normalized copy numbers have beensubmitted to GEO/NCBI data base (GSE28397).

Example 5 Meta-Analysis

Meta-analysis was performed on a public domain high density CGH dataseton 354 ovarian cancers (obtained from The Cancer Genome Atlas (TCGA)Project, located online at <cancergenome.nih.gov>) and 35 breast cancers(GSE15130). Array CGH data for all tumors imported into Nexus CopyNumber. Threshold for copy number loss was set to log2-0.5 (morestringent than the default setting of log2-0.2). Meta-analysis for geneexpression analysis was performed on a public domain gene expressiondataset (GSE22820) from the GEO/NCBI. Differential expression of TRPM3(the locus containing miR-204) in breast cancer samples was determinedby first performing RMA/quantile normalization and then comparing tonormal adjacent tissues within the same data set.

Example 6 Gene Expression Analysis

Human gene expression microarray data were generated using the AgilentHuman Whole Genome 4×44K array (Agilent Technologies). Total RNAisolated from HEK-293 cells transfected with miR-204 was co-hybridizedwith RNA from HEK-293 cell transfected with scrambled oligo (control)with dye-swap replicates, following the manufacturer's protocol.Relative gene expression ratios were extracted with Agilent's FeatureExtraction software (version 9.5.3.1, Agilent Technologies). Quantilenormalization was performed with the MATLAB Bioinformatics Toolbox(R2009a, Mathworks). To determine differential expression of genes, theStudent's t-test was applied to the normalized expression data.Signature gene sets were selected based on an FDR-adjusted P<0.05(Benjamini-Hochberg) and a fold change>2. Differential gene expressiondataset has been deposited in GEO/NCBI data base (GSE 28400).

Example 7 Plasmid Construction

For the pMIR-BDNF 3′ UTR construct, the 3′-UTR segment of the BDNF genewas amplified and sub-cloned downstream of the luciferase gene in thepMIR-REPORT vector (Ambion) at the HindIII and Spel sites. For thepSilencer-miR-204 construct, an approximately 500 by pri-miR-204 genomicsequence was amplified and cloned into the BamHI and HindIII sites ofthe pSilencer 4.1 Puro vector (Ambion).

Example 8 Cell Growth and Soft Agar Assay

Cell growth and soft agar colony formation assay using pSilencer-miR-204and pSilencerscramble (control) stable cell lines were performed asdescribed previously (Imam, et al., 2010).

Example 9 Tumorigenicity Assays in SCID Mice

Two million HEK-293 cells stably overexpressing either pSilencer-miR-204or pSilencer-scramble were injected into the renal capsules of 9RAG2^(−/−), γc^(−/−)SCID male mice (Taconic) and 9 control mice. Tumorvolume was assessed 24 d after transplantation, using the formulaπ/6×(L×D×W), where L is tumor length, D is depth and W is width. Sixmicrometer paraffin embedded tumor sections were stained withhematoxylin and eosin. All experimental procedures involving animalswere performed according to institutional ethical guidelines.

Example 10 Transwell Cell Migration and Basement Membrane MatrixInvasion Assay

Transwell cell migration assays were performed as previously described(Imam, et al., 2010). Invasion assays were performed with MDA-MB-231cells transfected with 75 nM miR-204 mimic or negative control mimic,then further transfected for 48 h with 75 nM miR-204-specific inhibitoror 25 ng of pBluescript KS⁺control, as described below. Forty-eighthours post-transfection, cells were seeded onto the inserts of 24-welltranswell plates precoated with Matrigel (1 mg/mL) (BD Biosciences).Serum-containing media and fibronectin (5 μg/mL) were added to the lowerchamber as chemoattractants. After 24 h incubation, cells on transwellinserts were washed, fixed, and non-invading cells and EC matrix weregently removed with a cotton swab. Invasive cells located on the lowerside of the chamber were stained with crystal violet (0.1%), air driedand photographed as described (Imam, et al., 2010).

Example 11 Therapeutic Experiments

100,000 MDA-MB-231-GFP-luc cells were injected into the tail vein.Starting from 7 d after tumor cell injection, hsa-miR-204 (n=6) orhsa-miR-204 mutant (n=6) oligos complexed with RNALancerII in vivodelivery formulation (Bioo Scientific) were injected every 5 d for 30 dat a rate of 1 mg of oligo per kg of body weight. All animals weresacrificed after the sixth injection. Lungs were fixed and analyzed formetastatic foci. Lung images were captured using fluorescence microscope(for GFP^(+ve) and luc^(+ve) foci). Livers were also harvested to assessmetastasis and hepatoxicity in hsa-miR-204 injected mice.

Example 12 Apoptosis Assays

Annexin V/PI staining on HEK 293 or HeLa cells transfected with 75 nMhsa-miR-204 mimic or negative control mimic was performed in triplicatewells using the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen)as described previously (32).

Example 13 Immunofluorescence

Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2%Triton X-100 for 15 min at room temperature (RT), then washed withphosphate-buffered saline (PBS) and blocked with 10% goat serum in PBSfor 45 min at RT. Cells were incubated with Rac1 mouse monoclonalantibody (ab33186, Abcam) diluted in PBS for 2 h at RT. Cells werewashed three times before incubating for 1 h at RT with 1:200 AlexaFluor 647 and Alexa Fluor 488 phalloidinconjugated secondary antibodies(A12379, Invitrogen, Molecular Probes). After three washes, cells weremounted on glass slides in Aqua-Poly/Mount medium containing DAPI(Polysciences). Photomicrographs were taken at 200×magnification on aNikon Eclipse TE2000-U microscope.

Example 14 Statistical Analysis

All values and error bars in graphs are mean±SEM; respective n valuesare indicated in figure legends; P-values are determined by two-tailedStudent's t-tests.

Example 15 miR-296-5p Inhibition Sensitizes Paclitaxel Response inTriple Negative Breast Cancers

Triple negative breast cancer cells were transfected with scramblecontrol or miR-296-5p inhibitor for 3-days followed by treatment witheither carrier or 8 nM Paclitaxel for two more days. Cell viability wascompared between carrier and drug treated miR-296-5p transfected cellsusing CellTiter-Glo (Promega Inc.). miR-296-5p inhibition significantlyreduced the cell viability in the presence of paclitaxel. Bar graphdepicts percentage of TNBC cell viability in control and miR-296-5ptransfected cells treated with vehicle control (DMSO) or paclitaxel (8nM). FIG. 13.

Example 16 miR-296-5p Inhibits Triple Negative Breast Cell Migration andInvasion

Triple negative breast cancer cells were transfected with scramblecontrol or miR-296-5p inhibitor for 48 hours. At 48 h aftertransfection, cells were trypsinized, resuspended in serum-free mediaand loaded into the top of 3 μm-pore-sized Transwell chambers (Corning,Corning, N.Y., USA). Serum-containing medium was placed in the bottomchamber as a chemoattractant and cells were incubated at 37° C. andallowed to migrate through the chemotaxis chamber for 24 h. The migratedcells at the bottom of the chamber were fixed with 10% formalin andstained with 0.4% crystal violet for 3 h. Experiments were repeated intriplicate and migrated cells were counted microscopically miR-296-5pinhibitor transfected cells had significantly lower migratory capabilitywhen compared to control transfected cells. Representative image showsinvaded cells. Picture was taken using Nikon microscope. FIG. 14.

Example 17 Efficacy of Treatment

To further establish the efficacy of these miRNAs in treating TNBC, weused ex-vivo tumor explant model. Freshly excised breast cancer tissueswere dissected into 1mm³ specimens, placed on hydrated gelatin sponge inexplant media supplemented with hydrocortisone and insulin at 37° C. inthe CO₂ incubator. Tumor explants from TNBC patients were treated withpaclitaxel and tissues were gently removed, fixed in 10% neutralbuffered formalin and processed for paraffin blocks. IHC analyses wereconducted to determine the Ki67 levels. IHC of surgical specimens andexplants reveal that explants retain the tissue architecture andproliferation properties of the original surgical specimen. Expectedly,paclitaxel treatment of explant revealed inhibition of Ki67 staining innon-relapsed TNBC, while advanced stage TNBC was not responsive to thepaclitaxel. FIG. 15.

Example 18 Liposome Formulation

Tumor explants growing on the gelatin sponge were treated with eithersensitizer miRNA (75 nM) alone or paclitaxel (1 μM) alone or with miRNA(75 nM) and paclitaxel (1 μM) together. Treatment of paclitaxel (byadding in explant media) alone for 48 hours resulted in significantinhibition of Ki67 levels (indicating tumor cell proliferation) inresponsive TNBC (early stage cancer). However, paclitaxel treatment didnot result in significant change in the Ki67 levels in relapsed TNBCexplants that are not responsive to paclitaxel Addition of sensitizermiRNA (75 nM) alone using liposome formulation for 48 hours resulted inmodest inhibition of Ki67 levels (indicating tumor cell proliferation)in tumor explants from advanced stage TNBC. However, the effect was muchmore robust when sensitizer miRNA (75 nM) was used in combination withpaclitaxel (1 μM). FIG. 16.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method for reducing growth of cancer cells comprising administeringan effective amount of a composition comprising hsa-miR-204 and/or ahomolog thereof to a subject having or suspected of having cancer cellsreduced in expression of hsa-miR-204 and/or a homolog thereof.
 2. Themethod of claim 1, wherein the cancer cells overexpress brain-derivedneurotrophic factor (BDNF) and/or ezrin.
 3. The method of claim 1,wherein the cancer cells are triple negative breast cancer (TNBC) cells,breast cancer cells other than TNBC cells, ovarian cancer cells, and/orpediatric renal tumor cells, or any combination thereof. 4.-5.(canceled)
 6. The method of claim 1, further comprising determining thelevel of hsa-miR-204 and/or a homolog thereof in the cancer cellswherein the level of hsa-miR-204 and/or a homolog thereof is reducedwhen compared to a control.
 7. (canceled)
 8. The method of claim 6,wherein the level of hsa-miR-204 and/or a homolog thereof in a bloodsample obtained from the subject is determined. 9.-12. (canceled) 13.The method of claim 1, wherein the subject is further administered acomposition comprising a second active agent before, after, and/orduring administration of the composition comprising hsa-miR-204 and/or ahomolog thereof.
 14. The method of claim 13, wherein the second activeagent comprises a cytotoxic chemotherapeutic, a nanobioconjugate, and/ora miRNA other than hsa-miR-204 and/or a homolog thereof, or anycombination thereof. 15.-20. (canceled)
 21. The method of claim 13,wherein cancer cells have become resistant to the second active agent.22.-27. (canceled)
 28. A method for identifying a subject likely tobenefit from therapy for reducing growth of cancer cells, the methodcomprising determining the level of hsa-miR-204 and/or a homolog thereofin cells of the subject, wherein the subject is determined as likely tobenefit from said therapy when the level of hsa-miR-204 and/or a homologthereof in the cells of the subject is reduced when compared to acontrol.
 29. (canceled)
 30. The method of claim 28, wherein the level ofhsa-miR-204 and/or a homolog thereof in a blood sample obtained from thesubject is determined.
 31. (canceled)
 32. A method for screening for apotential sensitizer and/or de-sensitizer miRNA that, when inhibited,changes the viability of cancer cell line cells cultured in a sub-lethalconcentration of a therapeutic active agent, the method comprising:culturing cancer cell line cells in a miRNA-inhibitor-containing culturecomprising a sub-lethal concentration of the therapeutic active agenttogether with an inhibitor of the candidate sensitizer miRNA; culturingcancer cell line cells in a miRNA-inhibitor-lacking culture comprisingthe sub-lethal concentration of the therapeutic active agent but lackingadded inhibitor of the candidate sensitizer miRNA; and determiningviability of the miRNA-inhibitor-containing cell population and themiRNA-inhibitor-lacking cell population after the period, wherein acandidate sensitizer miRNA is identified as a potential sensitizer miRNAif viability of the miRNA-inhibitor-containing cell population issignificantly higher than viability of the miRNA-inhibitor-lacking cellpopulation and wherein a candidate de-sensitizer miRNA is identified asa potential de-sensitizer miRNA if viability of themiRNA-inhibitor-containing cell population is significantly lower thanviability of the miRNA-inhibitor-lacking cell population.
 33. The methodof claim 32, wherein the potential sensitizer miRNA is hsa-miR-204,hsa-miR-185, hsa-miR-211, hsa-miR-367-5p, and/or hsa-miR-133A, and/or ahomolog thereof, or any combination thereof. 34.-35. (canceled)
 36. Themethod of claim 32, wherein the therapeutic active agent is paclitaxel.37. (canceled)
 38. The method of claim 32, wherein cancer cell linecells are TNBC cells.
 39. (canceled)
 40. The method of claim 32, whereinthe potential de-sensitizer miRNA is hsa-miR-129-3p, hsa-miR-296-5p,hsa-miR-216a, hsa-miR-1237, hsa-miR-1915*, and/or hsa-miR-320d, and/or ahomolog thereof, or any combination thereof. 41.-44. (canceled)
 45. Amethod for reducing growth of cancer cells comprising administering aneffective amount of a composition comprising a sensitizer miRNA and/or ahomolog thereof to a subject having or suspected of having cancer cellssignificantly reduced in expression of the sensitizer miRNA and/or ahomolog thereof.
 46. A method for reducing growth of cancer cellscomprising administering an effective amount of a composition comprisingan inhibitor of a de-sensitizer miRNA and/or homolog thereof to asubject having or suspected of having cancer cells significantlyincreased in expression of an inhibitor of a de-sensitizer miRNA and/orhomolog thereof.
 47. A method for identifying a subject likely tobenefit from therapy for reducing growth of cancer cells comprisingdetermining the level of a sensitizer miRNA, a sensitizer miRNA homolog,a de-sensitizer miRNA, and/or a de-sensitizer miRNA homolog, or anycombination thereof in cells of the subject.
 48. The method of claim 47,wherein the sensitizer miRNA is hsa-miR-204, hsa-miR-185, hsa-miR-211,hsa-miR-367-5p, and/or hsa-miR-133A, or a homolog thereof, or anycombination thereof.
 49. (canceled)
 50. The method of claim 47, whereinthe de sensitizer miRNA is hsa-miR-129-3p, hsa-miR-296-5p, hsa-miR-216a,hsa-miR-1237, hsa-miR-1915*, hsa-miR-320d, and/or a homolog thereof, orany combination thereof.